As a first step to studying the function of extremely large myofibrillar proteins using molecular genetic approaches, genes encoding these proteins must be isolated and cloned. In an attempt to isolate a clone carrying large portions of the coding sequence for mouse nebulin, a CDNA library was constructed using conditions designed to optimize the chances of cloning very large CDNAS. A putative nebulin CDNA clone has been isolated from this library. The clone carries an insert approximately 18 kilobases in length. Control experiments indicate that the entire insert in uncontaminated by the vector sequence, and hence must be genuine CDNA. We are currently mapping the insert for restriction endonuclease sites, and subcloning and sequencing selected segments. Several distinct mutations in the beta-myosin heavy chain (MHC) gene have been linked to hypertrophic cardiomyopathy (HCM), a serious genetic disease of the heart. Because the cardiac beta-MHC is also expressed in slow-twitch fibers of skeletal muscle, we have been able to study the mechanical properties associated with these myosin mutations in single skeletal muscle fibers obtained from HCM patients. We found that the normal and the mutant copy of the beta-MHC gene are on average equally expressed within single slow-twitch skeletal muscle fibers. Furthermore, we found that three distinct missense mutations in the beta-MHC gene that cause HCM produce either no change or only a small decrease in isometric force output of single slow-twitch skeletal muscle cells. In summary, these results demonstrate that isometric force production by myosin mutants need not be affected to cause HCM.